This invention, in general, refers to recombinant proteins suitable for the diagnosis of a porcine pathogen, and in particular, to recombinant fusion proteins which comprise the protein of the nucleocapsid of the porcine reproductive and respiratory syndrome virus (PRRSV), obtained both from European isolates and from American isolates, and its use in the diagnosis of PRRSV.
The causative agent of the porcine reproductive and respiratory syndrome (PRRS) is a new arterivirus (PRRSV) isolated for the first time in Europe [Wensvoort et al., Vet. Quarterly, 13, (1991), 121-130] and later in the United States [Collins et al., J. Vet. Diagn. Invest., 4, (1992), 117-126].
PRRSV causes an important pathology in the porcine livestock, characterised by reproductive disorders in the female pigs (miscarriages, piglets born dead or weak), and an increase of the perinatal mortality as well as dyspnea and piglet pneumonia [Terpstra et al., Vet. Quarterly, 13, (1991), 131-136).
PRRSV is an enveloped RNA virus, of approximately 62 nm of diameter, the viral genome of which comprises a single-stranded RNA of positive polarity of approximately 15 kilobases (kb) in length. The genome contains eight overlapping open reading frames (ORF) (Meulenberg et al., Virology, 192, (1993), 62-72). The virion contains 6 structural proteins encoded by ORFs 2 to 7 (Meulenberg et al., Virology, 206, (1995), 155-163; van Nieuwstadt et al., J. Virol., 70, (1996), 4767-4772]. The ORF7 encodes the nucleocapsid protein (protein N), which is the most immunogenic protein of PRRSV [Sanz et al., Second Symposium on Aujeszky and PRRS viruses, Copenhagen, Denmark (1995)], making it a good candidate for the detection of specific antibodies to the virus, and therefore, for the diagnosis of the disease.
Two different antigenic groups of PRRSV have been described, which correspond to the American and European isolates. The Japanese PRRSV isolates, as well as other Asiatic isolates are antigenicaly related with the American isolate. A characteristic example of a European PRRSV isolate is that deposited at the ECACC under the deposit number V93070108 [Spanish Patent ES 2.074.950], whereas a representative example of an American isolate of PRRSV is that identified as VR-2332 [European Patent EP 0 529 584]. The European and American isolates of PRRSV exhibit a different genotype [Meng et al., Archives of Virology, 140(1995), 745-755] with important antigenic differences [Wensvoort et al., J. Vet., Diagn. Inves., 4, (1992), 134138].
The serological differences between the European and American isolates complicate the diagnosis of the disease, as in many cases there is no cross-reactivity between the respective porcine sera (perhaps due to the absence of linear immunodominant epitopes). Additionally, the recent introduction in Europe of vaccines based on the American isolate represents an additional complication in the serological analysis of infected and/or vaccinated animals. Therefore, it is necessary to develop a diagnostic method which may allow to discriminate between both isolates, both in individual and in mixed populations, with the object of performing an accurate distinction with regard to the origin of the virus.
There are kits for the detection of the presence of PRRSV or of antibodies which recognise PRRSV, specific for the European and American isolates.
The usual methods for the diagnosis of PRRSV comprise carrying out an assay based on immunoperoxidase (IPMA) or the performance of an ELISA type assay based on pig""s lung alveolar macrophages or on antigenic recombinant proteins.
The techniques based on IPMA are expensive and bloody techniques, which require the use of pig""s lung alveolar ffacrophages coming from the sacrifice of animals, being it possible that said macrophages could be contaminated or infected with other pathogens [unless gnotobiotic or specific pathogen free (SPF) animals are used], and are not susceptible to automation as they require visual inspection under the microscope.
ELISA techniques based on macrophages require the use of macrophage extracts and therefore, present the same problems, in that sense, as the techniques based on IPMA. Additionally, the use of antigen in the form of a cell extract requires an internal reference with the same cell extracts, but without infection, in case there was any serum from an animal from the field which had natural reactivity, i.e. which had natural antibodies, which could mask the result.
The ELISA techniques based upon PRRSV recombinant proteins require the production of viral antigen in appropriate amounts and in an active form. The production of PRRSV antigens, for example the nucleocapsid protein, in tissue culture, is troublesome and expensive. On the other hand, the production of PRRSV recombinant proteins using recombinant baculovirus in permissive cells poses many problems, as the expression of said proteins in that system is costly and not very efficient, the proteins expressed are insoluble and only a small soluble fraction is recovered, which is the one used in the ELISA. Likewise, the products of ORF 3, 5 and 7 of PRRSV expressed by recombinant baculovirus in insect cells are difficult to purify. Additionally, reference uninfected insect cells must be included to rule out natural reactivity.
In general, diagnostic methods must be reliable, reproducible, sensitive, simple, cost-effective, of a wide spectrum and, advantageously, shall use active antigens the production of which, in unlimited amounts, is simple and cost-effective. Additionally, in the case of pathogens which have isolates with genetic, antigenic and pathogenic differences, it is convenient that they discriminate between the different isolates.
The currently available methods used for the diagnosis of PRRSV do not satisfactorily fulfill all the characteristics which have to be demanded from a diagnostic method and, therefore, there is still a need for other methods for the diagnosis of PRRSV which solve all or some of the problems mentioned above.
The invention provides recombinant fusion proteins, which comprise the nucleocapsid protein of PRRSV obtained both from European and American isolates, of use for the diagnosis of PRRSV. The invention also provides methods and kits for the diagnosis of PRRSV which comprise the use of said recombinant fusion proteins. The nucleic acid sequences which essentially encode said recombinant fusion proteins constitute an additional object of the present invention. The invention presented here proposes a new process for constructing the thermocouple.
The recombinant fusion proteins object of this invention comprise an amino acid sequence selected among the sequences identified (see the section regarding the LIST OF SEQUENCES) as SEQ. ID. No.: 1 and SEQ. ID. No.:2, or an active fragment of the same. The term xe2x80x9cactive fragmentxe2x80x9d, in the sense used in this description, refers to a protein fragment that is suitably recognised by positive sera against PRRSV, i.e. a fragment of ORF7 of PRRSV recognised in such a way that it allows the discrimination between PRRSV positive and negative sera.
SEQ. ID. No.: 1 shows the amino acid sequence corresponding to the nucleocapsid protein of a European isolate of PRRSV, specifically the one identified as Toledo 4/96 [Example 1], generally known as xe2x80x9cEuropean isolatexe2x80x9d in this description, while SEQ. ID. No.: 2 shows the amino acid sequence corresponding to the nucleocapsid protein of an American isolate, specifically the one identified as Canada 14/96 (Example 2), generally known as xe2x80x9camerican isolatexe2x80x9d in this description.
The recombinant fusion proteins also comprise an amino acid sequence, of variable length and composition, derived from the system chosen for the expression of the recombinant fusion protein, to which SEQ. ID. No.: 1, or SEQ. ID. No.: 2, or an active fragment of the same is fused, either directly, or through a binding section formed by a small number of amino acids derived from the genetic manipulation of the expression system for the recombinant fusion protein. Examples of said amino acid sequences include the full or partial gene product of gene 10 of phage T7, glutathione-S-transferase, 0-galactosidase and the product of the malE gene.
In a specific embodiment of this invention, the recombinant fusion proteins comprise an amino acid sequence chosen from SEQ. ID. No.: 1 and SEQ. ID. No.: 2, or an active fragment of the same, fused to a residue of 259 amino acids originating from the protein of gene 10 of phage T7, by means of a binding section of 4-6 amino acids. In a specific and preferred embodiment of this invention, the recombinant fusion proteins have the amino acid sequences identified as SEQ. ID. NO.: 8 or SEQ. ID. No.:9.
The recombinant fusion proteins provided by this invention can be obtained by expression of the sequence that encodes the nucleocapsid protein of PRRSV fused to the sequence that encodes another protein, or a part of it, as it happens, for example, in plasmids pET, pGEX, pEx, and pMal, which contain gene 10 of phage T7, the glutathione-S-transferase gene, the xcex2-galactosidase gene and the malE gene, respectively, in a suitable expression system such as a prokaryote, for example a micro-organism belonging to the genus Escherichia, Bacillus, Salmonella, Listeria, Yersinia, etc.
The sequence that encodes the nucleocapsid protein of PRRSV can be obtained from the viral RNA by conventional methods which comprise, for example, the synthesis of a copy DNA (cDNA) by reverse transcription and the enzymatic amplification of the nucleic acid fragment which contains the gene of ORF7 of PRRSV by means of the polymerase chain reaction (PCR) using-the suitable primers.
After this, the complete sequence that encodes the nucleocapsid protein of PRRSV is cloned into a plasmid which is used to transform appropriate host cells. Occasionally, the plasmid with the complete sequence of ORF7 of PRRSV is stabilised by cloning in a suitable cellular system, where the orientation and the size of the insert are also checked, and is subsequently cloned into the host cells suitable for the expression of the recombinant fusion protein. Any of the plasmids normally used in the transformation of prokaryotes can be employed.
The correct expression of the recombinant proteins was analysed by sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining or by immunoblotting, while its antigenicity was determined by an ELISA assay.
In a specific embodiment, the complete sequence of ORF7 of PRRSV, both of the European isolate and of the American isolate, has been cloned in plasmid pET3x, stabilised in E. coli DH5 cells, from where, having checked the orientation and size, it has been extracted and cloned in E. coli BL21 cells for the expression of the corresponding recombinant fusion protein.
The recombinant fusion proteins of this invention, containing the nucleocapsid protein of either the European or the American isolate of PRRSV, have been expressed in E. coli BL21 at very high expression levels when expressed as very large sized fusion proteins [45 or 46 kilodaltons (KDa)] (see Examples 3.4.1. and 4.4.1.). Previous attempts to express the nucleocapsid protein of PRRSV without fusing to other proteins or fragments of the same, or as small fusion proteins, did not allow the production of detectable levels of said protein. Although the recombinant fusion proteins provided by this invention produce inclusion bodies, said proteins can be easily recovered by solubilisation with guanidinium chloride. The recombinant proteins of this invention, with no additional manipulation, excepting a simple dilution in phosphate buffered saline (PBS), can be used to coat a suitable solid support prior to its use for diagnostic purposes.
The expression of the nucleocapsid protein of PRRSV in the form of a recombinant fusion protein in E. coli takes place at very high levels and it can be easily purified, hence making said protein an ideal candidate for the development of methods and kits for the diagnosis if PRRSV.
The recombinant fusion proteins provided by this invention are useful as a diagnostic reagent. This hypothesis is confirmed by the fact that monoclonal antibodies that compete efficiently with PRRSV-positive sera, only recognise the complete protein (Examples 3.4.2 and 4.4.2). Additionally, the most immunodominant epitope, the one recognised by monoclonal antibodies ICH5 and IDH10, is a conformation dependant epitope which requires the complete sequence of the nucleocapsid protein to be recognised.
The invention also provides a method for the diagnosis of PRRSV, in particular, a method for detecting the presence of antibodies which specifically recognise PRRSV, which comprises putting into contact a biological sample from an animal with a recombinant fusion protein provided by this invention, under conditions that allow the formation of an antigen-antibody complex, and detecting the antigen-antibody complex formed. In the sense used in this description, the term xe2x80x9cbiological samplexe2x80x9d refers to a sample coming from an animal, such as blood, serum, plasma, sputum, saliva or any other biological fluid, while the term xe2x80x9canimalxe2x80x9d includes all the mammals, and more specifically male pigs, female pigs and piglets.
The immunoassay can be, for instance, an indirect ELISA or a competitive ELISA. The detection of the antigen-antibody complex can be carried out by means of conventional techniques which include the production of a coloured reaction using, for example, an enzyme labelled reagent, such as peroxidase, and a suitable substrate.
Additionally, the invention provides a kit for the diagnosis of PRRSV, particularly, a kit for detecting the presence of antibodies which specifically recognise PRRSV in a biological sample from an animal, which includes a recombinant fusion protein provided by this invention and suitable means for detecting the antigen-antibody complex formed, which comprise the labelled reagent and the adequate substrate. The diagnostic kit may also contain a positive control and a negative control, with the object of facilitating the assessment of the results of the assay, by comparing against the results obtained with the standard sample.